Transcriptional Signature following Inhibition of Early-Stage Cell Wall Biosynthesis in Staphylococcus aureus

Abstract

To facilitate mode of action studies on antibacterial inhibitors of early-stage cell wall biosynthesis (CWB), we determined the transcriptional response of Staphylococcus aureus to depletion/inhibition of enzymes in this pathway by DNA microarray analysis. We identified a transcriptional signature distinct from that previously observed following exposure to inhibitors of late-stage CWB.

Defining the mode of action (MOA) of a new antibacterial agent is essential for guiding the further development process, including optimization of structure-activity relationships (3, 11). Transcriptional profiling using DNA microarrays has emerged as a powerful technique for MOA studies, since it can provide a genome-wide overview of the cellular response to antibacterial inhibitors at the level of transcription (3, 4, 11). By analyzing the genes deregulated following exposure to a novel antimicrobial, the MOA can be predicted by comparison with profiles obtained with established antibiotics with known MOAs (3, 4, 11).

Cell wall biosynthesis (CWB) is an important target for antibiotic action in Staphylococcus aureus (12). Substantial efforts have been directed toward the discovery of antibacterial inhibitors of early-stage CWB, mediated by the Mur enzymes (1, 2, 12) (Fig. 1). This portion of the pathway, also called stage I or the cytoplasmic stage of peptidoglycan synthesis (12), remains largely unexploited as a target for antibacterial chemotherapy (2, 12). To assist analysis of novel candidate anti-CWB inhibitors and to build on earlier studies that have identified transcriptional responses to antibiotics targeting the later stages of CWB (e.g., vancomycin, oxacillin) (7, 14), we sought to establish a universal transcriptional signature of S. aureus following inhibition of stage I CWB.

FIG. 1.

The stage I cell wall biosynthesis pathway in Staphylococcus aureus involves the biosynthesis of UDP-MurNAc-pentapeptide from UDP-GlcNAc, mediated by the Mur enzymes. The three points at which inhibition of the pathway was achieved in this study are shown in italics. PEP, phosphoenolpyruvate.

Unfortunately, there is a lack of characterized control inhibitors that specifically inhibit this stage of the pathway. Of the Mur enzyme inhibitors reported in the literature, only fosfomycin unequivocally mediates antibacterial activity specifically through inhibition of this portion of the pathway by interfering with the function of the UDP-N-acetylglucosamine enolpyruvyl transferase isoenzymes, MurA and MurZ (12). To circumvent this problem, in addition to using fosfomycin as a test inhibitor, we also employed genetic and posttranslational challenge to specifically inhibit/deplete the cell of other individual enzymes active in early-stage CWB (MurB and MurE).

S. aureus RN4220 and derivatives were used throughout this study. Strain TS2557 (8) carries a temperature-sensitive mutation in murB. S. aureus CYL368 (5) has been engineered to place murE under the control of the P_spac promoter, rendering expression of this gene conditional upon the presence of isopropyl-β-d-1-thiogalactopyranoside (IPTG). Since strain CYL368 required the presence of tetracycline in the growth medium to ensure maintenance of the lacI repressor plasmid (pMJ8246), pMJ8246 was also introduced into S. aureus RN4220 to enable both conditional and control strains to be cultured under identical conditions in the presence of tetracycline.

Strains were cultured in tryptone soya broth (TSB) with aeration. Conditional mutants were cultured under conditions that resulted in ca. 80% inhibition of growth in the mutant relative to the wild type, while drug-treated cultures were exposed to a concentration of antibiotic causing ca. 25% reduction in growth relative to untreated cultures after 40 min (3). Cells were harvested by adding 2 volumes of RNAprotect solution (Qiagen) directly to the culture and then processed according to the manufacturer's instructions.

S. aureus CYL368 and RN4220(pMJ8246) were grown overnight at 37°C in the presence of 3 μg tetracycline/ml and 0.3 mM IPTG. Cells were harvested, washed extensively to remove IPTG, and resuspended to an optical density at 600 nm (OD₆₀₀) of 0.05 in fresh broth lacking IPTG. Cultures were then grown at 37°C and harvested at an OD₆₀₀ of 0.25. Strains TS2557 and RN4220 were grown overnight at 30°C. Both strains were resuspended to an OD₆₀₀ of 0.075 in fresh broth, grown at 42°C, and harvested at an OD₆₀₀ of 0.25.

Fosfomycin treatment was conducted as follows. An overnight culture of S. aureus RN4220 grown at 37°C was used to inoculate fresh, prewarmed TSB to an OD₆₀₀ of ∼0.02 and grown at 37°C to an OD₆₀₀ of 0.1. The culture was split into prewarmed flasks, one of which contained fosfomycin (20 μg/ml), and incubation continued for 40 min before harvesting cells.

RNA was prepared using the RNA midi kit (Qiagen) from cells treated with lysostaphin. Control RNA and sample RNA were used to make differentially labeled cDNA by reverse transcription in the presence of fluorescent dyes Cy3 and Cy5. Both RNAs were then cohybridized, scanned, and analyzed as previously described (16). Cultures were grown in triplicate and hybridized in duplicate for a total of six arrays per condition. Microarray feature extraction was undertaken using ImaGene software (BioDiscovery), and the resulting fluorescent intensities were further processed using MAVI Pro software (MWG Biotech). Normalization and statistical analysis were performed using GeneSpring v7.3.1 software (Agilent Technologies). Differentially expressed genes for each condition were identified; these genes had normalized ratios that were >2-fold up- or downregulated with a P value of <0.05 by t test with Benjamini and Hochberg false discovery rate correction.

Genes subject to the same level of deregulation (≥2-fold up- or downregulated with a P of <0.05) under all three conditions (inhibition/depletion of MurA or MurZ, MurB, and MurE), were considered members of the transcriptional signature for inhibition of CWB (Table 1). This transcriptional signature primarily involved upregulation of genes involved in providing precursors essential for CWB (e.g., gltAB [glutamate biosynthesis], oppABCDF; [oligopeptide transport], dapABD, asd, thrBC, dhoM, ilvBCD, and leuABCD [amino acid biosynthesis]) and genes involved in the response to environmental stress (e.g., ctsR [transcriptional regulator of stress response], clpB [stress response-related chaperone], msrA2 [methionine sulfoxide reductase], and katA [catalase]) (Table 1).

TABLE 1.

Genes deregulated following inhibition/depletion of enzymes of stage I cell wall biosynthesis in S. aureus^a

Locus tag designation (S. aureus N315d)	Gene designation (S. aureus N315)	Fold change in expression following inhibition/depletion ofb:			Transcriptional response from previous studiesc
		MurA and MurZ	MurB	MurE	Conditional depletion of MurF	Inhibitors of stages II and III of CWB pathway
Upregulated genes
SA0011		3.47	3.09	6.69	U
SA0122	butA	3.67	5.39	9.02	U	U
SA0346		3.23	3.81	2.78		U
SA0422		2.72	3.10	3.60	U
SA0430	gltA	13.75	14.41	33.41	U	U
SA0431	gltB	7.74	10.09	11.18	U	U
SA0480	ctsR	2.13	5.13	2.73
SA0481		2.40	4.38	2.48		U
SA0482		2.37	4.83	2.30
SA0612		2.22	2.69	2.88	U
SA0707		3.19	2.96	5.18	U
SA0818	rocD	2.46	4.43	6.20	U
SA0835	clpB	4.05	10.40	3.39		U
SA0845	oppB	11.66	13.41	134.41	U	U
SA0846	oppC	12.64	13.17	149.31	U	U
SA0847	oppD	11.81	12.95	52.95	U	U
SA0848	oppF	13.48	17.55	118.00	U	U
SA0849	oppA	10.59	15.31	65.31	U	U
SA0871		3.40	3.59	3.45	U
SA0883		5.42	4.69	11.23	U
SA0886		2.50	3.40	2.96
SA0958		2.98	2.12	2.00	U
SA1019		2.57	2.45	3.25		U
SA1163		4.21	4.45	5.60		U
SA1164	dhoM	7.27	5.14	17.50	U	U
SA1165	thrC	7.58	5.17	12.94	U	U
SA1166	thrB	4.39	3.63	3.43	U	U
SA1170	katA	2.98	2.27	2.39
SA1226	asd	9.32	5.99	27.16	U	U
SA1227	dapA	8.40	5.58	17.73	U	U
SA1228	dapB	7.19	4.64	13.00	U	U
SA1229	dapD	5.96	4.26	10.58	U	U
SA1230		4.84	3.11	6.83	U	U
SA1231	dal	4.73	3.20	4.76	U
SA1235		2.09	2.01	2.02
SA1236		4.33	2.58	3.26	U	U
SA1238		3.88	2.62	2.96	U
SA1254		5.23	3.50	2.52		U
SA1255		9.90	3.80	2.83		U
SA1256		9.30	3.29	3.10		U
SA1257	msrA2	10.54	3.35	2.92		U
SA1532		4.58	3.05	7.69	U	U
SA1545	serA	6.24	6.00	6.67	U	U
SA1546		3.93	2.39	3.60	U	U
SA1691	sgtB	7.24	4.34	2.15	U	U
SA1858	ilvD	5.89	12.47	6.90	U
SA1859	ilvB	8.40	12.71	10.63	U
SA1860	ilvH	7.48	16.51	6.31	U
SA1861	ilvC	11.90	16.00	20.74	U	U
SA1862	leuA	9.05	14.46	12.70	U
SA1863	leuB	8.89	13.35	8.69	U
SA1864	leuC	8.92	14.99	10.40	U
SA1865	leuD	8.58	14.77	9.84	U
SA1866	ilvA	5.81	11.57	6.06	U
SA2112		2.75	2.16	3.10
SA2221		14.49	3.57	2.12		U
SA2235	opuCC	2.48	3.77	3.80	U
SA2236	opuCB	2.44	3.89	4.23	U
SA2237	opuCA	2.60	5.98	4.82	U
SA2240		2.15	2.40	3.38	U
SA2346		4.35	2.45	4.50		U
SA2347		4.51	2.95	5.68		U
SA2357		4.26	4.86	5.02	U	U
SA2396		3.05	4.08	4.99	U	U
SA2397		4.12	13.88	15.47	U	U
SA2473		2.56	3.43	3.56
SA2475		2.82	4.43	4.37	U	U
SA2476		3.76	5.61	7.00	U	U
SA2477		5.24	7.64	13.11
SA2478		6.19	8.12	19.76
SA2490		2.97	3.81	2.92		U
Downregulated genes
MW0552		0.49	0.46	0.33
SA0009	serS	0.49	0.26	0.34
SA0085		0.50	0.42	0.44		D
SA0100		0.21	0.09	0.20		U
SA0111	sirA	0.26	0.23	0.47	D
SA0183	glcA	0.17	0.23	0.29	D
SA0213		0.36	0.29	0.47
SA0268		0.31	0.36	0.18
SA0269		0.06	0.11	0.21
SA0270		0.20	0.38	0.28	D
SA0325	glpT	0.47	0.32	0.29
SA0423		0.36	0.29	0.31		D
SA0497	rplJ	0.47	0.28	0.27
SA0498	rplL	0.49	0.22	0.25	D
SA0654	fruB	0.18	0.34	0.34	U
SA0655	fruA	0.17	0.25	0.31
SA0820	glpQ	0.44	0.32	0.27	D
SA0905	atl	0.46	0.18	0.38		D
SA0912	qoxB	0.33	0.36	0.49		D
SA0913	qoxA	0.35	0.35	0.49		D
SA0951	potB	0.50	0.35	0.38	D
SA1056		0.22	0.49	0.25
SA1160	nucI	0.42	0.32	0.30	D
SA1502	rplT	0.46	0.21	0.23	D
SA1665		0.50	0.37	0.31	D	D
SA1850		0.43	0.37	0.48		U
SA1921	tdk	0.44	0.33	0.45	D
SA1979		0.35	0.32	0.40	D
SA2016	rpsI	0.49	0.35	0.40	D
SA2093	ssaA	0.24	0.05	0.09	D	D
SA2097	ssaA	0.37	0.05	0.08	D
SA2300		0.36	0.24	0.36	D
SA2332		0.37	0.19	0.16
SA2356	isaA	0.29	0.09	0.10		D
SA2430	aur	0.31	0.28	0.40	D
SA2486		0.49	0.36	0.39

^aGenes deregulated following inhibition/depletion of MurA and MurZ, MurB, MurE, and MurF, but not upon exposure to any inhibitors of stages II and III of CWB, are considered members of the universal transcriptional response to inhibition of stage I and are shown in bold type.

^bOnly those genes showing ≥2-fold deregulation in the same direction (up- or downregulation) under all three experimental conditions employed in this study (inhibition/depletion of MurA and MurZ, MurB, and MurE) are shown.

^cThe transcriptional responses of these genes in previous studies (following conditional depletion of MurF [13] or following inhibition of stages II and III of the CWB pathway [10]) are also shown. U, upregulated; D, downregulated.

^dExcept in cases where genes are not present or have not been designated in S. aureus N315. In this case, locus tag designations are from S. aureus MW2.

Little deregulation was detected in genes encoding enzymes directly involved in CWB or cell wall turnover, with the exception of upregulation of dal (alanine racemase), sgtB (penicillin-binding protein homologue), and downregulation of atl (autolysin) (Table 1). No consistent deregulation in expression of the Mur enzymes was observed, suggesting that as for Escherichia coli (9, 15), expression of genes involved in stage I peptidoglycan synthesis in S. aureus is constitutive, and CWB is unrestricted up to, and including, synthesis of the UDP-MurNAc pentapeptide.

We subsequently obtained a universal transcriptional signature specifically for inhibition of stage I CWB (Table 1). This was derived from our transcriptional signature for inhibition of CWB by including only those genes similarly deregulated following conditional depletion of MurF in a previous study (13) and subtracting all genes known to be deregulated following exposure to inhibitors of stage II/III CWB (bacitracin, vancomycin, oxacillin, and cefoxitin) (6, 7, 10, 14) (Table 1). Our results suggest that transcriptional profiling can be employed not only to identify inhibitors of CWB but also to establish whether they act on early or late stages in the biosynthetic pathway.

Microarray data accession number.

Fully annotated microarray data have been deposited in BμG@Sbase (accession number E-BUGS-71) (http://bugs.sgul.ac.uk/E-BUGS-71) and also ArrayExpress (accession number E-BUGS-71).